Tuftsin metallopeptide analogs and uses thereof

ABSTRACT

Tuftsin receptor-specific peptides, peptidomimetics and peptide-like constructs are provided, particularly for use in biological, pharmaceutical and radiopharmaceutical applications, in which the peptide, peptidomimetic or construct is conformationally fixed on complexation of the metal ion-binding portion thereof with a metal ion, resulting in a peptide, peptidomimetic or construct with increased affinity for the tuftsin receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority pursuant to 35 U.S.C. 119 basedupon Provisional Patent Application Serial No. 60/078,373, filed Mar.18, 1998 and No. 60/112,235 filed Dec. 14, 1998, the entire disclosuresof which are hereby incorporated by reference.

[0002] This application is also a continuation-in-part application ofU.S. patent application Ser. No. 08/6,60,697, entitled StructurallyDetermined Metallo-Constructs and Applications, filed Jun. 5, 1996,which in turn is a continuation-in-part application of U.S. patentapplication Ser. No. 08/476,652, entitled Peptide—Metal IonPharmaceutical Constructs and Applications, filed Jun. 7, 1995; theteachings of all of the foregoing applications are incorporated hereinby reference as if set forth in full.

GOVERNMENT RIGHTS

[0003] This invention was made in part with government support undergrant No. 1 R43 AI39343-01 from the U.S. Department of Health and HumanServices. The U.S. Government retains certain rights in the subjectinvention.

FIELD OF THE INVENTION

[0004] The present invention relates to tuftsin receptor-specificpeptide constructs which are conformationally fixed on complexation witha metal ion. The constructs, which may be peptidomimetic in nature, areuseful in pharmaceutical and radiopharmaceutical applications.

[0005] Throughout this application, various publications are referredto, each of which is hereby incorporated by reference in its entiretyinto this application to more fully describe the state of the art towhich the invention pertains.

BACKGROUND OF THE INVENTION

[0006] Tuftsin Receptor Peptide Construct. Ser. No. 08/660,697 teachescertain locally restricted peptides, in which the biological-functiondomain and metal-peptide backbone are combined, and thebiological-function domain is specific for the tuftsin receptor found onpolymorphonuclear (PMN) granulocytes, monocytes and macrophages.

[0007] Native tuftsin is a tetrapeptide of the sequence Thr-Lys-Pro-Arg,located as residues 289-292 of the Fc region of the heavy chain ofleukokinin (a cytophilic γ-globulin). It is liberated by a combinationof two cleavages. The C-terminal peptide bond is cleaved in the spleenby splenic enzyme and subsequent cleavage of the N-terminal peptide bondby enzyme leukokininase which occurs on the membranes of thegranulocytes where it acts to stimulate phagocytosis. The tuftsinsequence stimulates macrophages and polymorphonuclear granulocytestowards phagocytosis. This sequence thus has a role in the immune systemresponse for fighting infections and bacteria and other invasions. Thereare specific tuftsin receptors present on granulocytes and macrophages.The receptor density is approximately 50,000-100,000 per cell, with thereceptor-tuftsin complex reported to internalize after binding. Thus apeptide specific for the tuftsin receptor may be used in the treatmentof certain diseases, as is disclosed generally in U.S. Pat. No.4,390,528 to VA Najjar and U.S. Pat. No. 5,028,593 to K Nishioka, theteachings of which are incorporated herein by reference.

[0008] The '697 application teaches a precursor peptide, incorporatingboth a metal ion-binding backbone and a tuftsin receptor-specificbiological-function domain, which tuftsin receptor-specific domain isbiologically active only on labeling or complexing the metal ion-bindingbackbone with a metal ion, of the following general formula:

R₁-Aaa-Bbb-Ccc-Ddd-Eee-R₂

[0009] Where:

[0010] Aaa=L- or D-configuration residue selected from Thr, Cys, Pen,Pro, or Ser and corresponding des-amino derivatives.

[0011] Bbb=L- or D-configuration residue with a positively charged sidechain, and containing an N for metal ion complexation, such as Arg, Lys,Orn, homoArg, S-(2-aminoethyl)Cys, O-(2-aminoethyl)Ser and other similarbasic amino acids, and derivatives thereof

[0012] Ccc=L- or D-configuration residue with an uncharged side chain,and containing an N for metal ion complexation, such as Gly, Ala, Aib,Val, Nle, Leu and similar amino acids with un-charged side chains.

[0013] Ddd=L- or D-configuration residue, providing an S, and preferablyan S and N, for metal ion complexation, or alternatively two Ns formetal ion complexation, such as Cys, HomoCys, Pen, His and othersynthetic or derivatized amino acids.

[0014] Eee=L- or D-configuration residue with a positively charged sidechain, such as L- or D-isomers of Arg, Lys, Orn, homoArg,S-(2-aminoethyl)Cys, O-(2-aminoethyl)Ser and other similar basic aminoacids, and their corresponding des-carboxyl derivatives. A similaraliphatic or aromatic chain with a basic functional group can also besubstituted.

[0015] R₁=H, alkyl, aryl, alkylcarbonyl, arylcarbonyl, alkyloxycarbonyl,aryloxycarbonyl, or a polymer such as PEG, PVA, or polyamino acid,attached directly or through a carbonyl group. R₁ does not exist if Aaais a des-amino amino acid.

[0016] R₂=amide, substituted amide, ester, or a polymer such as PEG,PVA, or polyamino acid. R₂ does not exist if Eee is a des-carboxyl aminoacid.

[0017] One representative peptide from this series was the sequenceThr-D-Lys-Gly-D-Cys-Arg (34). This peptide displayed very high affinity(K_(D) =1-5 nM) for human leukocytes after its binding to reducedTcO[V]. When complexed to radioactive ^(99m)TcO[V], the peptidelocalizes to the site of inflammation or infection on i.v.administration. The affinity of the peptide which is not complexed to ametal ion is on the order of K_(D)=10⁻⁴ M.

[0018] The structure of the Thr-D-Lys-Gly-D-Cys-Arg peptide afterbinding to technetium is as follows:

[0019] The '697 application teaches that this peptide can similarly belabeled with Re, and that similar peptides can also be designed andsynthesized using an N₄ metal ion-binding domain, such asThr-D-Lys-Gly-D-His-Arg (36). Tuftsin receptor-specific peptidesdisclosed in '697 include Thr-D-Lys-Gly-D-Cys-Arg (34),Thr-D-Lys-Gly-D-His-Arg (36) and Pro-D.-Lys-Gly-D-Cys-Arg (35).

[0020] The peptides taught in '697 may be complexed with anon-radioactive ionic form of rhenium or another suitable isotope,thereby creating a non-radioactive metallopeptide drug for the treatmentof disease. Such peptides may also be radiolabeled with a diagnosticmetal ion, such as ^(99m)Tc, and used to determine sites ofconcentration of granulocytes and macrophages, such as infections andinflammations, or radiolabeled with a therapeutic metal ion, such as¹⁸⁶Re or ¹⁸⁸Re, and used in the treatment of disease.

[0021] In addition, tuftin has analgesic and other central nervoussystem effects. See, e.g., Herman et al., “Central Effects of Tuftsin,”in Antineoplastic, Immunogenic and Other Effects of the TetrapeptideTuftsin: a Natural Macrophage Activator, Najjar V A and Freidkin M,eds., New York Academy of Sciences, 1983 [hereinafter Antineoplastic],156-163; Paradowski et al., “The Influence of Tuftsin on Blood Pressurein Animals,” in Antineoplastic, 164-167; Fridkin and Najjar, Crit. Rev.Biochem. Med. Biol., 24 (1989). Herein disclosed are novel peptides andpeptidomimetics which are specific for the tuftsin receptor and may beused as an analgesic and in the treatment of various other centralnervous system conditions.

SUMMARY OF THE INVENTION

[0022] Metallopeptides. The present invention provides tuftsinreceptor-specific peptides which comprise a metal ion-binding backbonefor complexing with a metal ion, the peptide further comprising atuftsin receptor-specific biological-function domain, in which thetuftsin receptor-specific domain is conformationally constrained oncomplexing the metal ion-binding backbone with the metal ion. The metalion-binding backbone includes two or more contiguous amino acidsavailable for complexing with a metal ion, provided such that thepeptide is specific for the tuftsin receptor on complexing the metalion-binding backbone with a metal ion. The tuftsin receptor-specificdomain may be sychnological or rhegnylogical.

[0023] The present invention encompasses manufactured peptides andpharmaceutically acceptable salts thereof which are characterized byhaving a metal ion-binding backbone with two or more contiguous aminoacids available for complexing with a metal ion, and a tuftsinreceptor-specific biological-function domain which is conformationallyconstrained on complexing the metal ion-binding backbone with a metalion. In general, at least a portion of the peptide is conformationallyconstrained in a secondary structure on complexing the metal ion-bindingbackbone with the metal ion. The peptide may have a conformationallyconstrained global structure on complexing the metal ion-bindingbackbone with the metal ion. The tuftsin receptor-specific domain of thepeptide is substantially more potent on complexation of the metalion-binding backbone with the metal ion. The peptide is alsosubstantially more resistant to enzymatic degradation after complexingthe metal ion-binding backbone with a metal ion.

[0024] Typically, the metal ion-binding backbone is designed so that allof the valences of the metal ion are satisfied on complexation of themetal ion. In such instances, the metal ion-binding backbone may be aplurality of amino acids each containing at least one nitrogen, sulfuror oxygen atom available for complexing with the available valences ofthe metal ion. The metal ion-binding backbone also may include aderivatized amino acid or spacer sequence which contains at least onenitrogen, sulfur or oxygen atom available for complexing with theavailable valences of the metal ion.

[0025] The biological-function domain of the tuftsin receptor-specificmetallopeptide constitutes a ligand capable of binding with a receptor.The affinity of the tuftsin analog peptide ligand for its receptor willgenerally be substantially higher when the metal ion-binding backbone iscomplexed with the metal ion than that of the uncomplexed tuftsin analogligand.

[0026] The metal ion to be complexed may be selected from the group ofelements consisting of iron, cobalt, nickel, copper, zinc, manganese,arsenic, selenium, technetium, rathenium, palladium, silver, cadmium,indium, antimony, rhenium, osmium, iridium, platinum, gold, mercury,thallium, lead, bismuth, polonium and astatine. For the peptides of thisinvention, a metal ion which has a coordination number of 4 and is ableto complex with a tetradentate ligand is preferred. The isotope ^(99m)Tcis particularly applicable for use in diagnostic imaging, and theisotopes ¹⁸⁶Re and ¹⁸⁸Re are preferred for therapeutic applications.Non-radioactive rhenium is particularly applicable for use in makingnon-radioactive metallopeptides.

[0027] Tuftsin Analogs. Peptides of this invention may be manufacturedpeptides and pharmaceutically acceptable salts thereof containing ametal ion-binding backbone including two or more contiguous amino acidsavailable for complexing with a metal ion, and a biological-functiondomain specific for the tuftsin receptor, which tuftsinreceptor-specific domain is conformationally constrained on complexingthe metal ion-binding backbone with a metal ion.

[0028] The metal ion-binding backbone may be complexed with agamma-emitting metal ion, and the peptide used for diagnostic imaging ofsites of infection or inflammation. The peptide may also be used as animmunostimulatory agent, and may in such instances be complexed with ametal ion which is not radioactive. The foregoing peptides can becomplexed with technetium-99m (^(99m)Tc) a gamma emitter useful indiagnostic radioimaging, or with either radioactive or non-radioactiveisotopes of rhenium.

[0029] Accordingly, it is an object of this invention to devise,demonstrate and illustrate the preparation and use of highly specificconformationally restricted peptides, peptoids, related pseudopeptides,peptidomimetics and metallo-constructs formed by complexing sequencesthereof to a desired metal ion so that the topography of the side chainsin the resulting complex is a biologically active three-dimensionalstructure which binds to a tuftsin receptor.

[0030] Another object of this invention is to provide tuftsinreceptor-specific peptide-metal ion complexes which have a higher levelof stability and are less susceptible to proteolysis than either theuncomplexed peptide, or other peptides known in the art.

[0031] Another object of this invention is to provide for tuftsinreceptor-specific peptide analogs which are not conformationallyrestricted in the absence of a metal ion, whereby the uncomplexedpeptide analog is either inactive or demonstrates low potency, but whichhave high potency and concomitant conformational restriction oncomplexation with a metal ion.

[0032] Another object of this invention is to utilize metal complexationin a tuftsin receptor-specific peptide to cause specific regionalconformational restrictions in the peptide so that the peptideconformation at the metal binding site is conformationally fixed onmetal complexation.

[0033] Another object of this invention is to complex a tuftsinreceptor-specific peptide to a metal ion so as to alter the in vivobiodistribution profile, rate and mode of clearance from the body,bioavailability and pharmacokinetics in mammals.

[0034] Another object of this invention is to provide tuftsinreceptor-specific peptide-metal ion complexes which utilize stablenon-radioactive metal ions, with the biological-function domain havingspecific tuftsin-like biological activity, such as for therapeutictreatment of disease.

[0035] Another object of this invention is to provide a molecule which,on complexing with a metal ion, includes a biological-function domainwhich is specific for tuftsin receptors, and which stimulatespolymorphonuclear granulocytes, monocytes and macrophages towardsphagocytosis, and may be used in diagnostic methods for abscess andinfection imaging.

[0036] Another object of this invention is to provide a peptide-metalion complex with a region specific for the tuftsin receptor onpolymorphonuclear granulocytes and macrophages which increases theantigenic profile of antigens presented to such polymorphonucleargranulocytes and macrophages, thereby resulting in production of highertiter antibodies.

[0037] Another object of this invention is to develop a tuftsinreceptor-specific peptide-metal ion complex which is an antagonist oftuftsin.

[0038] Another object of this invention is to develop a tuftsinreceptor-specific peptide-metal ion complex which is an agonist oftuftsin.

[0039] Another object of this invention is to complex tuftsinreceptor-specific peptides with radiometal ions for use in whole bodyimaging and radiotherapy so that the resulting peptide-metal ion complexis of higher affinity and specificity for the tuftsin receptor than theuncomplexed peptide molecule, and the resulting radiolabeled species isessentially carrier-free in terms of tuftsin receptor recognition.

[0040] Another object of this invention is to provide tuftsinreceptor-specific peptide-metal ion complexes which can transit thegut-blood barrier, without significant enzymatic or peptidasedegradation, and may be adapted for oral administration.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Tuftsin receptor-specific peptide-metal ion complexes, and theprecursor uncomplexed sequences, which include peptide, peptidomimetic,peptide-like and metallo-constructs, are provided for biological,pharmaceutical and radiopharmaceutical applications. In the tuftsinreceptor-specific peptide-metal ion complexes the construct isconformationally fixed, with the tuftsin receptor-specific domaingenerally having increased affinity for its target on labeling the metalion-binding backbone with a metal ion.

[0042] The peptide constructs of this invention can include a metal ion,and for embodiments in which the metal ion is used diagnostically ortherapeutically, a medically useful metal ion. The metal ion isoptionally radioactive, paramagnetic or superparamagnetic. The metal ionis an ionic form of an element selected from the group consisting ofiron, cobalt, nickel, copper, zinc, manganese, arsenic, selenium,technetium, ruthenium, palladium, silver, cadmium, indium, antimony,rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead,bismuth, polonium and astatine. The metal ion may also be an ionicradionuclide of indium, gold, silver, mercury, technetium, rhenium, tin,astatine or copper.

[0043] A radioactive medically useful metal ion may generate gamma rays,beta particles, or positrons which are converted into gamma rays oncollision with electrons. The medically useful metal ion may be used indiagnostic imaging procedures including gamma scintigraphy, specificphoton emission computerized tomography, or positron emissiontomography. The medically useful metal ion may also be useddiagnostically in magnetic resonance imaging. Medically useful metalions may also be used therapeutically.

[0044] The type of medically useful metal ion depends on the specificmedical application. Particularly useful metal ions include elements25-30 (Mn, Fe, Co, Ni, Cu, Zn), 33-34 (As, Se), 42-50 (Mo, Tc, Ru, Rh,Pd, Ag, Cd, In, Sn) and 75-85 (Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Po,At). Isotopes of the elements Tc, Re, and Cu are particularly applicablefor use in diagnostic imaging and radiotherapy. The isotope ^(99m)Tc isparticularly applicable for use in diagnostic imaging. Otherradionuclides with diagnostic or therapeutic applications include ⁶²CU,⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰⁹Pd ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰³Pb ²¹¹Pb and²¹²Bi.

[0045] The tuftsin receptor-specific domain of the peptide is a sequenceof one or more amino acids which constitute a biologically activepeptide sequence, exhibiting binding to the tuftsin receptor found oncells, tissues or organs. The tuftsin receptor-specific domain alsoincludes any sequence which may be consecutive amino acids(sychnological) or may be non-consecutive amino acids (rhegnylogical),of one or more amino acids which forms a tuftsin receptor-specificligand, which ligand is capable of forming a specific interaction withits acceptor or receptor. The term “receptor” is intended to includeboth acceptors and receptors. The peptide or the biological-functiondomain may optionally transmit a signal to the cells, tissues or othermaterials associated with the biological receptor after binding. Thetuftsin receptor-specific domain may thus be either an agonist orantagonist, or a mixed agonist-antagonist. The tuftsin receptor-specificdomain may also constitute a member of a “specific binding pair,”wherein a specific binding pair comprises at least two differentmolecules, where one molecule has an area on the surface or in a cavitywhich specifically binds to a particular spatial and polar organizationof the other molecule.

[0046] Radiopharmaceutical Applications. Products of this invention maybe employed as radiopharmaceutical agents. For example, when labeledwith gamma-emitting radioisotopes, such as ^(99m)Tc, the products may beutilized as a diagnostic agent in nuclear medicine.

[0047] Products of this invention may also be used as therapeutic agentswhen labeled with alpha- or beta-emitting radioisotopes. For example,peptides labeled with alpha- or beta-emitting radioisotopes, such asrhenium-186 (¹⁸⁶Re) or rhenium-188 (¹⁸⁸Re), can be used for treatingdiseases.

[0048] For radiopharmaceutical applications, and other medicalapplications, the products of this invention offer significantadvantages over conventional linear or single-chain peptide constructs.For example, it is known that conformationally constrained and dimericpeptides derived from hypervariable loop sequences of antibodies canbind antigens with an affinity up to 40-fold higher than that obtainedwith linear sequence peptides. The peptides of this invention areconformationally constrained on labeling with a metal ion, and have ahigher affinity than that obtained with conventional linear sequences.

[0049] For radiopharmaceutical and other medical applications, thepeptides of this invention may be delivered to a subject by any meansknown in the art. This includes intravenous injection, subcutaneousinjection, administration through mucous membranes, oral administration,dermal administration, regional administration to an organ, cavity orregion, and the like.

[0050] Non-Radiopharmaceutical Therapeutic Applications. The products ofthis invention may be used for therapeutic applications, and areparticularly useful for peptide drugs in which a tuftsinreceptor-specific biological-function domain is required. In theseapplications, the metal ion may serve only to conformationally constrainthe peptide, or a portion thereof, or may itself be related to thetherapeutic nature of the agent.

[0051] Specific Tuftsin Analogs. The peptides of Table 1 weresynthesized by solid-phase peptide synthesis using Boc-chemistry, andwere purified by HPLC to purity levels of 95% or higher and analyzed byelectrospray mass spectrometry. For all products, the experimental andcalculated molecular masses were identical. TABLE 1 Primary structureand designation of tufisin ana- logs, shown uncomplexed to a metal ion.Designation Primary Structure 1 Thr-Lys-GIy-D-Cys-Arg 2Ac-His-Asn-Aln-Lys-Thr-D-Lys-Gly-D-Cys- Arg 3 D-Lys-GIy-D-Cys-Arg 4Thr-D-Lys-D-Ser-Cys-Arg 5 His-Asn-D-Ala-Lys-Thr-D-Lys-Gly-D-Cys- Arg 6PEG₅₀₀₀-D-Lys-Gly-D-Cys-Arg 7 His-Asn-D-Ala-Lys-Pro-D-Lys-Gly-D-Cys- Arg8 Arg-D-Arg-GIy-D-Cys-Arg 9 Thr-D-Arg-Gly-D-Cys-Arg 10Pro-D-Arg-Gly-D-Cys-Arg 12 Lys-Thr-D-Arg-Gly-D-Cys-Arg 13Gly-D-Lys-D-Cys-Arg 14 Thr-D-Lys-D-Cys-Arg 15 Thr-D-Arg-Gly-D-Cys-Lys 16Thr-D-Orn-Gly-D-Cys-Arg 17 Thr-D-Arg-D-Lys-D-Cys-Arg 18Gly-D-Arg-D-Cys-Arg 19 D-Arg-D-Lys-D-Cys-Arg 20 D-Arg-Arg-D-Cys-Arg 21D-Arg-Lys-D-Cys-Arg 22 Thr-Arg-Arg-Cys-Arg 23 Arg-Gly-Gly-D-Cys-Leu-Arg24 Arg-Thr-Gly-D-Cys-Arg 25 Thr-D-Arg-Gly-Cys-Arg 26Arg-Gly-Gly-D-Cys-Arg 27 Thr-D-Gln-Gly-D-Cys-Arg 28Thr-Arg-Gly-D-Cys-Arg 29 Thr-Arg-Gly-Gly-D-Cys-Arg 30Thr-D-Arg-Gly-D-Cys-Orn 37 Ac-D-Lys-Gly-D-Cys-Arg 38Thr-D-Lys-Lys-D-Cys-Arg 39 Lys-Thr-D-Arg-Lys-D-Cys-Arg 40Thr-D-Lys-Arg-D-Cys-Arg 41 Thr-Arg-Arg-D-Cys-Arg 42Thr-D-Lys-Orn-D-Cys-Arg 43 Lys-Thr-D-Arg-D-Lys-D-Cys-Arg 44Thr-D-Arg-D-Arg-Cys-Arg 45 Thr-Arg-D-Lys-Cys-Arg 46Thr-Lys-D-Lys-Cys-Arg 47 Thr-D-Arg-Gly-D-Cys-Arg 48Thr-D-Arg-Gly-Cys-Arg 49 Thr-Arg-Gly-D-Cys-Arg 50Thr-D-Lys-Gly-D-Cys-Nle 51 Thr-D-Ala-Gly-D-Cys-Arg 52Ala-D-Lys-Gly-D-Cys-Arg 53 Lys-Thr-D-Lys-Ser-D-Cys-Arg 54Lys-Thr-D-Arg-Ser-D-Cys-Arg 55 Thr-Lys-Pro-Pro-Arg-[NH-(CH₂)₆-CO]-Thr-D-Lys-Gly-D-Cys-Arg 56 Thr-D-Lys-Gly-D-Cys-Arg-[NH-(CH₂)₆-CO]-Thr-Lys-Pro-Pro-Arg 57 Thr-D-Lys-Ser-D-Cys-Arg 58Thr-D-Ser-Ser-D-Cys-Arg 59 Thr-D-Ser-Ser-D-Cys-Ser

[0052] The peptides of Table 1 are synthesized by any means known in theart, including those methods disclosed in '697, and are evaluated todetermine their ability to complex ^(99m)Tc. It was determined that eachpeptide complexed ^(99m)Tc very effectively. Each peptide was labeledusing an identical protocol. A 5-10 μg sample of the peptide taken in0.001 N aq. HCl was mixed with 1-30 mCi of generator-eluted Na^(99m)TcO₄in a 5 ml serum vial. The volume of the resulting mixture was adjustedto 600 μl using injectable saline. A 400 μl volume of a freshly preparedand nitrogen-purged phthalate-tartrate-Sn(II) buffer (40:10:1 mM) wasthen added to the vial under a nitrogen head space. The vial wasimmediately sealed and placed in a shielded boiling water bath. After 15min. the vial was removed from the water bath and allowed to come toroom temperature. A small amount of the sample (1-10 mCi) was analyzedby reverse-phase HPLC using a C-18 column (VYDAC, 250×4.8 mm, 10 micronparticle size) with a 0-30% acetonitrile gradient in 0.1% aq. TFAcompleted in 30 min. at a flow rate of 1.5 ml/min. Radioelution profileswere generated using an in-line radioactivity detector (Beckman, Model170). The Tc-peptide complexes were usually obtained as a mixturecharacterized by two HPLC peaks, presumptively due to syn- andanti-isomerism in the Tc=O core. The HPLC profiles for each of the^(99m)Tc-peptides showed a complete absence of free, uncomplexed^(99m)Tc (which elutes at 2.5-3 min. under the reverse-phase HPLCconditions described). The radiochemical purity, as calculated from theHPLC profiles, ranged from 90-97%.

[0053] The peptides of Table 1 may alternatively be labeled with^(99m)Tc by any of the means taught in '697, including use ofstannous-tartrate-succinate buffer, stannous-EDTA-succinate buffer,stannous stabilized in glucoheptonate, or a stannous-borate-tartratebuffer, as well as other means of labeling with ^(99m)Tc known in theart.

[0054] The peptides of Table 1 may be complexed with non-radioactivemetal ions, and rhenium is a preferred ion. Peptides in solution may belabeled by treatment with the rhenium transfer agent ReO[V]Cl₃(PPh₃)₂ inthe presence of 1,8-Diazabicyclo[5,4,0] unclec-7-ene as a base. Metalcomplexation in the presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene as abase can conveniently be accomplished at ambient room temperature. In analternative method of metal complexation a mild base, such as sodiumacetate, can be used. In this case the peptide is taken in a suitablesolvent, such as DMF, NMP, MeOH, DCM or a mixture thereof, and heated to60-70° C. with the rhenium transfer agent ReO[V]Cl₃(PPh₃)₂ in thepresence of sodium acetate for 15 minutes. Similarly, other bases suchas triethylamine, ammonium hydroxide and so on, may be employed. Variousmixtures of the solvents, also in combination with MeOH, and DCM, CHCl₃and so on, may also be employed to yield optimized complexation results.

[0055] The peptides of Table 1 may be used as diagnostic imaging agentsfor localizing sites of infection or inflammation, particularly whenlabeled with ^(99m)Tc, or as immunotherapeutic agents, particularly whenlabeled with radioactive isotopes of rhenium or complexed withnon-radioactive isotopes of rhenium, all as described elsewhere hereinand in '697.

[0056] The peptides of Table 1 have, on labeling with technetium,^(99m)Tc or a similar metal ion, a core configuration as shown below. Inthis configuration, each of R₁, R₂, R₃ and R₄, if provided, may be oneor more amino acids as described herein, or may be other constructs asdescribed herein. Amino acids, if provided, at R₂, R₃ and R₄, may format least a part of the tuftsin receptor-specific biological-functiondomain. Amino acids with cationic side chains at two or more of R₂, R₃or R₄ may form at least a part of the tuftsin receptor-specificbiological-function domain. Peptides of this invention with amino acidshaving cationic side chains at R₂, R₃ and R₄ include: 17, 19, 20, 21 and22. Peptides with cationic side chains at R₂ and R₄ include: 34, 35, 36,37, 32, 33, 4, 5, 7, 8, 9, 11, 10, 12, 15, 16, 25, 28, and 30. Peptideswith cationic side chains at R₃ and R₄ include: 13, 14 and 18.

[0057] Peptides as disclosed herein may include either a D- orL-cysteine residue in the metal ion-binding domain and may incorporate acore sequence described by the formula:

[0058] R₂-R₃-Cys-R₄

[0059] wherein R₂ is D- or L- Lys, Arg, Gly, Thr, Gln or Orn

[0060] R₃ is D- or L- Gly, Ser, Lys, Arg

[0061] Cys is D-Cys or L-Cys and

[0062] R₄ is D- or L- Arg, Lys, Leu or Orn The invention is furtherillustrated by the following non-limiting examples.

EXAMPLE 1 Kit Formulations for One Step Labeling with ^(99m)Tc

[0063] The peptides 34 and 35 were formulated in lyophilized, one-step^(99m)Tc-labeling kit form. This was done to demonstrate that thepeptides could be formulated in a commercially appropriate format. Fiftyto 100 vials were prepared by making a bulk nitrogen-purged solutionobtained by mixing the peptide (5-10 μg per vial) with 400,μl volume ofa freshly prepared and nitrogen-purged phthalate-tartrate-Sn(II)] buffer(40:10:1 mM, pH 6.0) per vial, all under sterile conditions. Theresulting solution was filtered through 0.22 m low-binding filter anddispensed (400 μl per vial) in pyrogen-burned 5 ml serum vials. Thevials were frozen, lyophilized and sealed under inert gas. The kits werestored at 4° C. until used. To label, 1-25 mCi of ^(99m)Tc asNa^(99m)TcO₄ in 0.5-4 ml saline was added to the vial, and the vialplaced in a boiling water bath for 15 min. The complexation efficiencywas analyzed by RP-HPLC as described above. Lyophilized formulationsyielded comparable results to those described above, ranging from 90-97%radiochemical purity.

EXAMPLE 2 SepPak Analysis of ^(99m)Tc-Labeled Peptides

[0064] C-18 SepPak cartridges (Millipore Inc, Bedford, Mass.) wereemployed for quick analysis of labeling efficiency. This system providesthree quantitative measures: (a) unbound ^(99m)Tc in the form of^(99m)Tc-NaTcO₄ and ^(99m)Tc-tartrate, (b) peptide-bound ^(99m)Tc, and(c) ^(99m)Tc-colloid. A freshly prepared ^(99m)Tc-peptide complex(50-200 μCi in 10-200 μl) was loaded on a pre-conditioned cartridge.Pre-conditioning was done by successively eluting the cartridge withethanol (10 ml) and 0.001 N aq. HCl (10 ml). The Tc-peptide-loadedcartridge was serially eluted with a 10 ml solution of 0.001 N aq HCl,10% aq. EtOH, and 100% EtOH. All three eluants were counted forradioactivity in a dose calibrator, and the cartridge itself was alsocounted. The 0.001 N HCl eluant yielded the estimate of free,uncomplexed ^(99m)Tc. The second eluant, 10% aq. EtOH, eluted all of the^(99m)Tc-bound peptide. The column radioactivity representednon-elutable ^(99m)Tc-colloid. The radioactivity in these threefractions was computed in terms of percentages. The results with thepeptides of Table 1 showed 1-3% free ^(99m)Tc, 90-95% peptide-bound^(99m)Tc, and 2-5% ^(99m)Tc-colloid. These results, together with HPLCprofiles, indicated very high labeling efficiency for the peptides ofTable 1.

EXAMPLE 3 Stability of ^(99m)Tc-Peptide Complexes as Measured byCysteine Challenge Studies

[0065] The sulfhydryl group of Cys complexes to Tc with high affinity.Cys challenge studies therefore provide an estimate of the relative bondstrength of Tc-peptide complexes. A constant amount of freshly labeled^(99m)Tc-peptide (10-100 μCi in 10-100 μl) was incubated with increasingamounts (0-100 mM) of Cys in PBS (pH 7.4) at 37° C. for one hour. Eachsample was then analyzed either by HPLC or by the SepPak techniquesdescribed earlier. The amount of radioactivity not complexed to thepeptide and the fraction remaining complexed to the peptide werecomputed. A graph of these values against Cys concentration wasconstructed to yield an IC₅₀ value (Cys concentration required to remove50% of peptide-bound ^(99m)Tc). The IC₅₀ values for peptide 34 and 35ranged from 50-75 mM. Based on similar studies on variousradiopharmaceuticals described in the literature, these valuesdemonstrate very good stability for these Tc-peptide complexes. Bycomparison, the in vivo concentration of free sulfhydryl groups in serumis approximately 0.6 mM.

EXAMPLE 4 Metabolic Stability of ^(99m)Tc-Peptides in vivo in Rodents

[0066] The Tc-labeled peptides 1 through 36 were tested for in vivometabolic stability in mice. Freshly labeled ^(99m)Tc-peptide wasinjected through the tail vein in mice (50 μCi) or rats (100 μCi). Theurine from these animals was collected after 30 and 120 min. The urinesamples were centrifuged to remove any particulates. A urine samplecontaining 1-10 μCi (in 50-200 μl volume) was analyzed by RP-HPLC forthe integrity of the labeled peptide. In all cases the ^(99m)Tc-elutionprofile of the peptide in urine was similar to the original^(99m)Tc-peptide, revealing no change in the peptide as a result ofinjection into rodents. All the peptides were, therefore, metabolicallystable and excreted intact in the urine. ^(99m)Tc-labeled 34 wasadministered both by subcutaneous injection and orally in mice, and theurine analysis by HPLC of these animals for both routes ofadministration also revealed 100% intact Tc-peptide, indicating oralabsorption and complete in vivo metabolic stability of the peptide.

EXAMPLE 5 Metabolic Stability of ^(99m)Tc-Peptides in Human Plasma invitro

[0067] The peptides 35 and NH₂-Thr-D-Lys-Gly-D-Cys-Arg-COOH as describedin '697 were assayed by incubating a sample of ^(99m)Tc-peptide (100 μCiin 100 μl) with 100 μl of freshly prepared human plasma at 37° C. for1-2 hrs. The sample was then injected into an RP-HPLC column asdescribed above. Comparisons of the radio-elution profile with that ofthe original Tc-labeled peptide showed no change in the peptide,indicating metabolic stability of Tc-peptides in the presence of humanplasma proteases.

EXAMPLE 6 Affinity of Tc-Labeled Peptides to Human PolymorphonuclearGranulocytes and Cultured HL-60 Cells

[0068] The peptides 3 and 34 were used in direct saturation bindingstudies. Both peptides exhibited saturation binding kinetics. The assayswere performed by both filtration and centrifugation techniques.Tc-labeled 34 bound HL-60 and PMN granulocytes in saturable fashion witha K_(D) value of 1-5 μM. Tc-labeled 3 exhibited a K_(D) value of 5-15nM. In these experiments various amounts of the unlabeled peptides wereused (10-100 μg) and labeled with a constant amount of ^(99m)Tc andsimilar K_(D) values were obtained. These results supported thehypothesis that Tc-labeled molecules are the only biologically relevantspecies and the presence of varied amounts of unlabeled peptides have noeffect on receptor binding.

[0069] Competitive binding studies were performed to establish (a)binding of Tc-labeled peptide to the tuftsin receptor, (b) receptoraffinity and (c) lack of receptor affinity of unlabeled peptides.^(99m)Tc-labeled peptide (a gamma emitter) was competed separately withincreasing concentrations of ⁹⁹Tc-labeled peptide (a weak beta emitter)and natural tuftsin. The IC₅₀ value for ⁹⁹Tc-labeled 34 was between 1-5nM (similar to the values obtained for ^(99m)Tc-labeled 34 in saturationbinding experiments). Natural tuftsin exhibited an IC₅₀ of approximately100 mM. The unlabeled peptide (peptide uncomplexed to Tc) was 2000-3000fold less potent than the Tc-labeled counterpart. Tc-labeled 34 is up to100-fold more potent than natural tuftsin, and may be the most potenttuftsin molecule known so far.

EXAMPLE 7 Stimulation of Phagocytic Activity of Granulocytes by^(99m)Tc-Peptides

[0070] Phagocytic assays using freshly harvested PMNs were performedusing ⁹⁹Tc-labeled or corresponding Re-labeled peptides according to themethod described by Fridkin et al. (Biochim Biophys Acta, 1977, 496,203-211). Metal ion-complexed 34 was identified as a potent agonist instimulating phagocytosis of heat inactivated yeast cells. The doseresponse curve was bell shaped as observed for other tuftsin peptidesreported in the literature. Maximal effect was observed at 1-5 nMconcentrations of the labeled peptide which paralleled its affinity inPMN binding experiments. Metal ion-complexed 3 and 35 were identified aspotent antagonists in inhibiting tuftsin or Re-34 induced phagocytosis.3, 34 and 35 uncomplexed with a metal ion were not biologically relevantat the concentrations at which the metal ion complex species werepotent.

EXAMPLE 8 Abscess Localization Biodistribution, and Clearance Studies inRodents

[0071] Normal mice and rats were used for biodistribution studies. Inmost of these studies the animals were injected 24 hrs prior to thestudy with turpentine (50 μl per mouse and 100 μl per rat) in the leftthigh region to cause an experimentally induced sterile abscess. Mice(female, 25 g average weight) were injected with 50 μCi of the^(99m)Tc-peptide and rats (female, 230 g average weight) ?were given 100μCi, all through tail veins. The biodistribution studies were performedat 30 and 120 min. time points. Animals were sacrificed and selectedorgans dissected and weighed, and associated radioactivity measured. Theabscessed muscle as well as normal contralateral muscle was alsoexcised. The data was computed using a computer program custom-designedfor ^(99m)Tc-labeled preparations. With all the ^(99m)Tc-peptides nomajor accumulation of radioactivity in any organ was observed except forkidney and liver. Spleen and bone marrow in some cases also hadaccumulations slightly above the background levels established for otherorgans. The major excretion route for these peptides was through kidneyand liver. As much as 75% of the radioactivity was excreted within thefirst 30 min. In the case of liver excretion, some radioactivity wasfound in the gut at the 120 min. time point. Table 2 (mouse model) andTable 3 (rat model) summarizes the results of the abscess uptake forvarious peptides studied during this study period. It is evident fromthese tables that peptides with high localization in the abscess as wellwith abscess to muscle ratios of as high as 10:1 were obtained TABLE 2Abscess localization of the ^(99m)Tc-peptide in mice model. Percentinjected dose in whole blood and abscessed muscle is shown. Also shownare the ratios of the abscess (Ab) to blood (Bl) and to normal muscle(Mu). 30 Min. 120 Min. Total Total ^(99m)Tc- Blood Abscess Ratio RatioBlood Abscess Ratio Ratio Peptide (% ID) (% ID) Ab:Bl Ab:Mu (% ID) (%ID) Ab:Bl Ab:Mu 34 0.49 0.31 1.45 6.59 0.45 0.30 1.84 10.81 35 5.48 1.700.62 3.15 1.22 0.36 0.63 4.63 1 6.75 2.07 0.73 3.05 1.05 0.44 1.08 7.192 3.84 1.28 0.78 4.54 0.61 0.36 1.31 8.25 3 3.18 1.58 1.83 3.53 0.320.50 3.61 8.38 4 7.70 1.54 0.44 3.78 1.60 0.44 0.64 4.91 5 6.78 1.180.40 2.55 2.00 0.62 0.64 6.03 7 15.29 1.83 0.29 3.21 3.76 0.43 0.27 3.288 31.28 1.10 0.10 2.12 4.25 0.38 0.25 2.51 9 15.3 1.14 0.20 2.97 8.360.98 0.28 3.1 11 3.35 1.25 0.87 8.89 1.04 0.33 0.77 6.01 12 3.35 1.801.31 5.45 0.81 0.35 1.53 8.90 13 3.34 2.00 1.38 6.42 0.38 0.49 2.8312.03 17 3.48 1.05 0.68 2.70 0.66 0.34 1.15 6.09 20 3.19 1.00 0.79 3.321.20 0.21 0.56 4.76 21 2.61 1.19 1.04 3.86 0.34 0.16 1.08 3.67 22 3.361.54 0.78 8.59 0.55 0.24 0.77 2.79

[0072] TABLE 3 Abscess localization of the ^(99m)Tc-peptide in ratmodel. Percent injected dose in whole blood and abscessed muscle isshown. Also shown are the ratios of the abscess (Ab) to blood (Bl,) andto normal muscle (Mu). 30 Min. 120 Min. Total Total ^(99m)Tc- BloodAbscess Ratio Ratio Blood Abscess Ratio Ratio Peptide (% ID) (% ID)Ab:Bl Ab:Mu (% ID) (% ID) Ab:Bl Ab:Mu 34 4.70 0.21 0.56 3.04 0.37 0.041.35 4.87 3 8.06 0.35 0.79 3.51 0.96 0.12 2.22 10.48 4 6.19 0.21 0.572.76 2.95 0.04 0.60 4.35 9 4.15 0.18 0.56 3.66 1.34 0.07 0.63 4.84 116.56 0.38 0.85 4.48 0.85 0.12 1.48 8.43 10 5.71 0.34 0.81 3.82 0.62 0.091.95 7.07 12 6.58 0.36 0.77 5.47 1.11 0.12 1.45 9.05 13 5.50 0.24 0.824.02 0.77 0.06 1.23 7.09 14 4.61 0.28 0.75 4.15 0.57 0.05 1.09 5.86 156.51 0.27 0.68 4.25 0.98 0.08 1.31 9.77 16 6.44 0.26 0.69 4.20 0.84 0.101.53 9.13 18 5.29 0.28 0.87 3.61 0.96 0.08 1.48 9.18 19 5.29 0.24 0.844.28 0.87 0.06 1.36 6.45

EXAMPLE 9 Preparation of Analogs Conjugated to Higher Molecular WeightMolecules

[0073] The conjugation of high molecular weight carrier molecules, suchas PEG, PVA, fatty acids and others, to the peptides of Table 1 isachieved either after the synthesis of the peptide or during thesynthesis of the peptide. PEG of various molecular weights (100-8000)and mono-methoxy PEG of similar molecular weights may be used byactivation with disuccinimide carbonate as taught by S. Zalipsky(Bioconjugate Chemistry 4:296-299, 1993). The activated PEG is thentreated with the peptide taken in phosphate buffer (125 mM, pH 6.5) inpresence of 1 mM HOBt. After 1 hour at room temperature, the reactionmixture is extracted several times with dichloromethane. The combinedorganic extract is washed with water and evaporated to dryness. Theproduct is then precipitated by the addition of anhydrous ether, andpurified by precipitation from an ethanol-ether system. Alternativelycarrier molecules are attached to the peptide during its synthesis bysolid-phase or solution-phase methods of peptide synthesis. The carriermolecules are attached either at the N-terminus or C-terminus, or atboth termini.

[0074] 6 was synthesized by solid phase methods of peptide synthesisusing monoethoxy-PEG-carboxylate of an average molecular weight of 5,000for N-terminal conjugation to the peptide at the final step ofsynthesis.

EXAMPLE 10 Imaging of Sites of Infection or Inflammation

[0075] Any of the tuftsin analogs of Table 1 are radiolabeled asdescribed, and optionally by means of the lyophilized kit of Example 1or modifications thereof, with between 5 and 20 mCi of ^(99m)Tc. Thepeptides of this invention may be employed at a ratio of peptide tometal ion of as low as 2:1, and in some instances lower, and thus theminimum quantity of peptide is generally determined by the quantity ofmetal ion. Generally, the total amount of peptide for diagnostic imagingapplications will be between about 1 and 10 μg. The labeled tuftsinanalog is administered, by i.v. injection or orally, to patientssuspected of having one or more sites of abscess, infection orinflammation, and periodic whole body scintigraphic images are obtainedfollowing administration to determine the localization of theradiolabeled peptide to the abscess, infection or inflammation site orsites. The effectiveness of the labeled peptide to image the site orsites of abscess, infection or inflammation is noted.

EXAMPLE 11 Therapeutic Treatment with Rhenium Labeled Analogs

[0076] Any of the tuftsin analogs of Table 1 are complexed with an ionicform of non-radioactive rhenium. In one method, any of the tuftsinanalogs of Table 1 may be complexed with an ionic form ofnon-radioactived rhenium by treatment in solution with the rheniumtransfer agent ReO[V]Cl₃(PPh₃)₂ in the presence of1,8-Diazabicyclo[5,4,0] undec-7-ene as abase. Metal complexation in thepresence of 1,8-Diazabicyclo[5,4,0]undec-7-ene as a base canconveniently be accomplished at ambient room temperature.

[0077] Following complexation, the metallopeptides may be convenientlystored by any means known in the art, including lyophilization, freezingor retention in solution atappropriate storage temperatures.Alternatively, and depending on the disease to be treated and theselected mode of administration, the metallopeptides may be compoundedinto a tablet, capsule, caplet, syrup or other similar oraladministration formulation, or alternatively, may be compounded into anymethod or system employed for the administration of peptides andpeptide-based drugs, including intravenous formulations, intramuscularformulations, aerosol formulations, transmucosal formulations,transdermal formulations, nasal absorption formulations, oral cavityabsorption formulations and the like. Depending on the disease orcondition to be treated, which may be any of various immune systemdisorders or other conditions for which an immunostimulatory agent isappropriate, or may be any condition for which an analgesic agent isappropriate, including various central nervous system conditions, aneffective amount of the metallopeptide is administered on an appropriateschedule.

[0078] All of the foregoing examples are merely illustrative, and otherequivalent embodiments are possible and contemplated as within the scopeof the invention. Variations and modifications of the present inventionwill be obvious to those skilled in the art. It is intended that theappended claims encompass all such modifications and equivalents.

What is claimed is:
 1. A peptide comprising a metal ion-binding backboneincluding two or more contiguous amino acids available for complexingwith a metal ion, which peptide is specific for the tuftsin receptor oncomplexing the metal ion-binding backbone with a metal ion.
 2. A peptideof claim 1 having a sequence selected from the group consisting of:Thr-Lys-Gly-D-Cys-Arg, Ac-His-Asn-Ala-Lys-Thr-D-Lys-Gly-D-Cys-Arg,D-Lys-Gly-D-Cys-Arg, Tbr-D-Lys-D-Ser-Cys-Arg,His-Asn-D-Ala-Lys-Thr-D-Lys-Gly-D-Cys-Arg,His-Asn-D-Ala-Lys-Pro-D-Lys-Gly-D-Cys-Arg, Arg-D-Arg-Gly-D-Cys-Arg,Thr-D-Arg-Gly-D-Cys-Arg, Pro-D-Arg-Gly-D-Cys-Arg,Lys-Thr-D-Arg-Gly-D-CyS-Arg, Gly-D-Lys-D-Cys-Arg, Thr-D-Lys-D-Cys-Arg,Thr-D-Arg-Gly-D-Cys-Lys, Thr-D-Orn-Gly-D-Cys-Arg,Thr-D-Arg-D-Lys-D-Cys-Arg, Gly-D-Arg-D-Cys-Arg, D-Arg-D-Lys-D-Cys-Arg,D-Arg-Arg-D-Cys-Arg, D-Arg-Lys-D-Cys-Arg, Thr-Arg-Arg-Cys-Arg,Arg-Gly-Gly-D-Cys-Leu-Arg, Arg-Thr-Gly-D-Cys-Arg, Thr-D-Arg-Gly-Cys-Arg,Arg-Gly-Gly-D-Cys-Arg, Thr-D-Gln-Gly-D-Cys-Arg, Thr-Arg-Gly-D-Cys-Arg,Thr-Arg-Gly-Gly-D-Cys-Arg, Thr-D-Arg-Gly-D-Cys-Orn,Ac-D-Lys-Gly-D-Cys-Arg, Thr-D-Lys-Lys-D-Cys-Arg,Lys-Thr-D-Arg-Lys-Lys-Cys-Atg, Thr-D-Lys-Arg-D-Cys-Arg,Thr-Arg-Arg-D-Cys-Arg, Thr-D-Lys-Orn-D-Cys-Arg,Lys-Thr-D-Arg-D-Lys-D-Cys-Atg, Thr-D-Arg-D-Arg-Cys-Arg,Thr-Arg-D-Lys-Cys-Arg, Thr-Lys-D-Lys-Cys-Arg, Thr-D-Arg-Gly-D-Cys-Arg,Thr-D-Arg-Gly-Cys-Arg, Thr-Arg-Gly-D-Cys-Arg, Thr-D-Lys-Gly-D-Cys-Nle,Thr-D-Ala-Gly-D-Cys-Arg, Ala-D-Lys-Gly-D-Cys-Arg,Lys-Thr-D-Lys-Ser-D-Cys-Arg, Lys-Thr-D-Arg-Ser-D-Cys-Arg,Thr-D-Lys-Ser-D-Cys-Arg, Thr-D-Ser-Ser-D-Cys-Arg, andThir-D-Ser-Ser-D-Cys-Ser.


3. A pharmaceutically acceptable salt of any of the peptides of claim 1.4. A peptide of claim 1, wherein the metal ion-binding backbone of thepeptide is complexed with a metal ion.
 5. A peptide of claim 4, whereinthe metal ion is radioactive
 6. A peptide of claim 4, wherein the metalion is an isotope selected from the group consisting of isotopes oftechnetium and rhemnum
 7. A peptide of claim 4, wherein the peptidecomplexed with a metal ion is substantially resistent to enzymaticdegradation
 8. A peptide of claim 2, wherein the affinity of the peptidefor the tuftsin receptor is substantially higher when the metalion-binding backbone is complexed with the metal ion than is theaffinity of the peptide for the tuftsin receptor when the metalion-binding backbone is not complexed with the metal ion.
 9. A peptideof claim 2, wherein the peptide is conjugated to a high molecular weightcarrier
 10. A method of imaging a site of infection or inflammation in amammal comprising administering a diagnostically effective amount of acomposition comprising a peptide in accord with claim 2, the peptidebeing in a form complexed with a diagnostically useful metal ion
 11. Themethod of claim 10 wherein the diagnostically useful metal ion is^(99m)Tc
 12. A method of causing an immunostimulatory response in amammal comprising administering art effective amount of a compositioncomprising a peptide in accord with claim 2, the peptide being in a formcomplexed with a non-radioactive metal ion.
 14. The method of claim 12wherein the non-radioactive metal ion is a form of rhenium
 15. A tuftsinreceptor-specific antagonist peptide comprising the sequenceD-Lys-Gly-D-Cys-Arg complexed with a metal ion
 16. A manufacturedpeptide and pharmaceutically acceptable salts thereof which is specificfor the tuftsin receptor upon complexing with a metal ion, said peptidebeing of the general formula: R₂-R₃-Cys-R₄ wherein R₂ comprises D- or L-Lys, Arg, Gly, Thr, Gln or Orn; wherein R₃ is D- or L- Gly, Ser, Lys,Arg; wherein Cys is D-Cys or L-Cys; and wherein R₄ comprises D- or L-Arg, Lys, Leu or Orn
 17. A peptide of claim 16 having a sequenceselected from the group consisting of: Thr-Lys-Gly-D-Cys-Arg,Ac-His-Asn-Ala-Lys-Thr-D-Lys-Gly-D-Cys-Arg, D-Lys-Gly-D-Cys-Arg,Thr-D-Lys-D-Ser-Cys-Arg, His-Asn-D-Ala-Lys-Thr-D-Lys-Gly-D-Cys-Arg,His-Asn-D-Ala-Lys-Pro-D-Lys-Gly-D-Cys-Arg, Arg-D-Arg-Gly-D-Cys-Arg,Thr-D-Arg-Gly-D-Cys-Arg, Pro-D-Arg-Gly-D-Cys-Arg,Lys-Thr-D-Arg-Gly-D-Cys-Arg, Gly-D-Lys-D-Cys-Arg, Thr-D-Lys-D-Cys-Arg,Thr-D-Arg-Gly-D-Cys-Lys, Thr-D-Orn-Gly-D-Cys-Arg,Thr-D-Arg-D-Lys-D-Cys-Arg, Gly-D-Arg-D-Cys-Arg, D-Arg-D-Lys-D-Cys-Arg,D-Arg-Arg-D-Cys-Arg, D-Arg-Lys-D-Cys-Arg, Thr-Arg-Arg-Cys-Arg,Arg-Gly-Gly-D-Cys-Leu-Arg, Arg-Thr-Gly-D-Cys-Arg, Thr-D-Arg-Gly-Cys-Arg,Arg-Gly-Gly-D-Cys-Arg, Thr-D-Gln-Gly-D-Cys-Arg, Thr-Arg-Gly-D-Cys-Arg,Thr-Arg-Gly-Gly-D-Cys-Arg, Thr-D-Arg-Gly-D-Cys-Orn,Ac-D-Lys-Gly-D-Cys-Arg, Thr-D-Lys-Lys-D-Cys-Arg,Lys-Thr-D-Arg-Lys-D-Cys-Arg, Thr-D-Lys-Arg-D-Cys-Arg,Thr-Arg-Arg-D-Cys-Arg, Thr-Lys-D-Lys-Cys-Arg, Thr-D-Arg-D-Arg-Cys-Arg,Thr-Arg-D-Lys-Cys-Arg, Thr-Lys-D-Lys-Cys-Arg, Thr-D-Arg-Gly-D-Cys-Arg,Thr-D-Arg-Gly-Cys-Arg, Thr-Arg-Gly-D-Cys-Arg, Thr-D-Lys-Gly-D-Cys-Nle,Ala-D-Lys-Gly-D-Cys-Arg, Lys-Thr-D-Lys-Ser-D-Cys-Arg,Lys-Thr-D-Arg-Ser-D-Cys-Arg, Thr-D-Lys-Ser-D-Cys-Arg,Thr-Lys-Pro-Pro-Arg-[NH-(CH₂)₆-CO]-Thr-D-Lys-Gly-D-Cys-Arg, andThr-D-Lys-Gly-D-Cys-Arg-[NH-(CH₂)₆-CO]-Thr-Lys-Pro-Pro-Arg


18. A peptide of claim 16, wherein the peptide is complexed with a metalion.
 19. A peptide of claim 18, wherein the metal ion is radioactive 20.A peptide of claim 18, wherein the metal ion is an isotope selected fromthe group consisting of isotopes of technetium and rhenium
 21. A peptideof claim 18, wherein the peptide complexed with a metal ion issubstantially resistent to enzymatic degradation
 22. A peptide of claim17, wherein the affinity of the peptide for the tuftsin receptor issubstantially higher when the metal ion-binding backbone is complexedwith the metal ion than is the affinity of the peptide for the tuftsinreceptor when the metal ion-binding backbone is not complexed with themetal ion.
 23. A peptide of claim 17, wherein the peptide is conjugatedto a high molecular weight carrier.